Genetically
engineered pacemakers could be a possible alternative to implantable
electronic devices for the treatment of bradyarrhythmias. The
strategies include upregulation of beta adrenergic receptors,
conversion of myocytes into pacemaker cells and stem cell therapy.
Pacemaker activity in adult ventricular myocytes is normally repressed
by the inward rectifier potassium current (IK1). The IK1
current is encoded by the Kir2 gene family. Use of a negative construct
that suppresses current when expressed with wild-type Kir2.1 is an
experimental approach for genesis of genetic pacemaker.
Hyperpolarisation activated cyclic nucleotide gated (HCN) channels
which generate If current, the pacemaker current of heart can be
delivered to heart by using stem cell therapy approach and viral
vectors. The unresolved issues include longevity and stability of
pacemaker genes, limitations involved in adenoviral and stem cell
therapy and creation of genetic pacemakers which can compete with the
electronic units.

Keywords: Gene therapy, Pacemaker current, HCN channels.

Introduction

Implantable
electronic pacemakers remain the treatment of choice for high degree
atrioventricular blocks and sinus node dysfunction. The shortcomings of
electronic pacemakers include limited battery life, need for lead
implantation into heart and lack of response to autonomic and
physiologic demands on the heart. Molecular approaches to the
development of a biological pacemaker are a conceptually attractive
alternate treatment modality for heart blocks. The approaches attempted
to provide such pacemaker function include up regulation of β2
adrenergic receptors1, down
regulation of K+ current IK12
and over expression of HCN2 (hyperpolarisation activated cyclic
nucleotide gated) channels the molecular correlate of the endogenous
cardiac pacemaker current If3.
The genetic treatment can be applied to heart by plasmid injection, use
of viral constructs or stem cell therapy4,5.

Molecular targets for genesis of
biological pacemaker

β2 adrenergic
receptors

The sinus node
has a higher density of β adrenergic receptors (βAR) compared with
surrounding atrium1. This
density of βAR and its regulation of If current suggest that increases
in the density of βAR in the vicinity of the sinus node may lead to an
increase in heart rate. The up regulation of β2
adrenergic receptors can be achieved by plasmid injection into heart.
It was noted that after injection of plasmids in porcine right atrium
heart rates were 50% faster than those of controls. One potential
limitation of this strategy is that the diseased endogenous cardiac
pacemaker mechanisms are left intact and the β2 receptor is
used as a nonspecific stimulator of heart rate. It can influence other
catecholamine sensitive channels also.

HCN channel
and If current
Action potential of pacing cells is unique in that
they have a slow depolarizing phase, rendering them spontaneously
active6. The depolarization
involves interaction between HCN
channels and L & T type calcium channels. The modification of these
channels is a therapeutic target.
HCN channels
generate If current which contribute to genesis of pacemaker
activity. If channel is activated on membrane
hyperpolarisation rather than on depolarization7. It has four fold selectivity
for K+ than Na+. The typical features of If current include
activation by hyperpolarized membrane potential, conduction of Na+ and
K+, modulation by cyclic adenosine monophospate (CAMP) and blockade by
cesium (Cs+)8. HCN generated
current also has the above features. Four different HCN genes have been
identified9. HCN1 is the
most rapidly acting channel, HCN4 the slowest with HCN2 and 3
possessing intermediate kinetics10.
HCN1, 2 and 4 have been found to express in adult heart, HCN4 being the
most highly expressed one in SA node. HCN2 expression was noted in
atrium, ventricle and SA node.
HCN can be
delivered to heart by adenoviral construct or using stem cells. The
nucleic acids delivered by adenoviruses do not integrate into genome as
they are episomal. Stem cell therapy may be more promising than viral
strategy. The approach using HCN may be less problematic and
proarrythmic as it incorporates the endogenous pacemaker channel gene,
which selectively activates only during diastole14.

Inward
Rectifier Potassium Current (IK1)
IK1
and other background K+ selective currents contribute to action
potential depolarization and establish diastolic resting membrane
potential. Down regulation of the background K+ current IK1
is one of the approaches attempted to provide pacemaker function.
Genetic suppression of IK1 can converts quiescent
myocytes
into pacemaker cells.
IK1
is the classical inward rectifier potassium current. Inwardly
rectifying K+ channels (Kir) are responsible for stabilizing the
resting membrane potential. Inward rectification is a phenomenon in
which conductance of a Kir channel increases with hyperpolarisation but
decreases with depolarization. Rectification in Kir channels results
from voltage dependent channel block by intracellular cations12. IK1 is absent
or poorly
expressed in sinus and AV nodal cells. Native IK1
in human
ventricular myocytes is reduced by adrenergic receptor stimulation.
It was
observed that a dominant negative strategy to reduce IK1,
which usually maintain ventricular myocytes at negative membrane
potentials, induced spontaneous impulse initiation in guinea pig heart.
The inward rectifier potassium current is encoded by Kir2 gene family.
Replacement of 3 amino acid residues in the pore structure of Kir2.1
creates a dominant negative construct12.
Downregulation of IK1 removes an important
determinant of
repolarisation leading to prolonged repolarisation in cells lacking
this current13. This may
result in excessive dispersion of repolarisation leading to theoretical
risk of proarrhythmia.

Stem cell therapy

Human
embryonic stem cells can be used to create pacemakers or adult
mesenchymal stem cells may be used as platforms for delivery of
pacemaker genes to myocardium. The advantage of these cells includes
their ability to make functional gap junctions and generate spontaneous
rhythms15. The approach
using embryonic stem cells carry the problems of identifying
appropriate cell lineages, possibility of stem cell differentiation
into lines other than pacemaker cells, and potential for neoplasia.
Adult mesenchymal stem cells are biologically inert vectors which can
deliver genetic information to myocardium. Human mesenchymal stem cells
(hMSCs) as a platform for delivery of genes into heart is a more
attractive option because they can be obtained in large numbers, easily
expanded in culture, capable of long term transgene expression and
their administration can be autologous or via banked stores15.

Gene therapy versus stem cell
therapy

In gene
therapy a cardiac myocyte is converted into a pacemaker cell whereas in
stem cell therapy myocytes retain their original function. An inherent
problem of gene therapy is use of viruses. Replication deficient
adenoviruses with little infectious potential lead to only transient
improvement in pacemaker function. Retroviruses may be carcinogenic and
infective.

Important studies on biological
pacemakers

1. Molecular
transfer of the human β2 Adrenergic receptor cDNA
Effects of
transferring the human β2 adrenergic receptor were studied
by Edelberg JM et al1 in
chronotropy studies with isolated myocytes, and transplanted as well as
endogenous murine heart. Murine embryonic cardiac myocytes were
transiently transfected with plasmid constructs. The total percentage
of spontaneously contracting myocytes was greater in β2AR
transfected cells compared with controls. Also the percentage of
myocytes with chronotropic rates more than 60 beats per minute was
greater in β2AR population than controls. To study the ex
vivo effects of targeted expression of β2AR a murine
neonatal cardiac transplantation model was used. Injection of β2AR
construct increased the heart rate by 40%. These studies demonstrate
that local targeting of gene expression may be a feasible modality to
regulate the cardiac pacemaking activity.

2. Local
expression of HCN2 in canine left atrium
Research by
Jihong Qu et al13 showed
that HCN2 over expression provides an If - based pacemaker
current
sufficient to drive the heart when injected into a localized region of
atrium. Adenoviral constructs of mouse HCN2 and green fluorescent
protein (GFP) or GFP alone were injected into LA, terminal studies
performed 3-4 days later, myocytes examined for native and expressed
pacemaker current (If). Spontaneous LA rhythms occurred
after vagal stimulation-induced sinus arrest in 4 of 4 HCN2 + GFP dogs
and 0 of 3 GFP dogs (P<0.05).

3. Biological
pacemaker implanted in canine left bundle branch
Alexi N.
Plotnikov et al14 studied
the effect of administration of the HCN2 gene to the left bundle branch
system of dogs. An adenoviral construct incorporating HCN2 and green
fluorescent protein (GFP) as a marker was injected via catheter under
fluoroscopic control into the posterior division of the LBB. Controls
were injected with an adenoviral construct of GFP alone or saline.
During vagal stimulation, HCN2 injected dogs showed rhythms originating
from the left ventricle, the rate of which was significantly more rapid
than controls.

4. Human
mesenchymal Stem Cells as a gene delivery system to create cardiac
pacemaker
Potapova I et
al3 tested the ability of
human mesenchymal stem cells to deliver a biological pacemaker to the
heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by
electroporation expressed current as If - like. They
demonstrated that genetically modified hMSCs can express functional
HCN2 channels in vitro and in vivo, mimicking over exression of HCN2
genes in cardiac myocytes, and represent a noval delivery system for
pacemaker genes into the heart or other electrical syncytia.

Limitations of approaches to
development of biological pacemaker

Use of viruses
to deliver the necessary genes has inherent problems. Replication
deficient adenoviruses that have little infectious potential lead to
only transient improvement in pacemaker function as well as
potential inflammatory responses. Retroviruses carry a risk of
carcinogenicity and infectivity. Limitations of stem cell therapy
include immunogenicity of cell, the potential for neoplasia, proper
engineering of pure cardiac lineages and spatial non uniformity of
implants. Regulating the level of expression to achieve optimal
pacemaker rate is critical. Biological pacemaker needs an optimal cell
mass and optimal cell-cell coupling for long term normal function.
Research is ongoing to identify optimal cell numbers and coupling
ratios needed to optimize the function of biological pacemakers.
A major issue
is duration of efficacy of biological pacemakers. The duration of
pacemaker function in approaches using viruses depend on how long the
viruses and resulting protein constructs survive in the host. To ensure
long term function the appropriate delivery system in which the
construct is effective for long periods must be identified. What will
be the longevity and stability of next generation of pacemaker genes?
The onset of
pacemaker function after a pause following the last intrinsic beat is a
critical factor. Can a pacemaker gene inserted into proximal conduction
system create a functioning biological pacemaker which can drive the
ventricle in demand mode when the sinus node signal fails? This
requires proper engineering of genes. Considering the cell-cell
coupling differences in gene therapy and stem cell therapy, the
engineering of mutant genes will differ importantly between approaches.

The autonomic
responsiveness of biological pacemakers, the ideal site for
implantation, the extent of recovery of diseased sinus node and the
ideal construct to be preferred remain unanswered questions. None of
the studies tested whether a biological pacemaker could be engineered
into the ventricular conducting system. Will the functional
characteristics of biological pacemakers compete with that of
electronic units available?